Download Rajiv Gandhi University of Health Sciences, Karnataka Bangalore

RajivGandhiUniversity of Health Sciences
Bangalore
Master of Dental Surgery (MDS)
Periodontology
AECS Maaruti College of Dental Sciences and Research Centre,
Bangalore - 76
Rajiv Gandhi University of Health Sciences, Karnataka
Bangalore
ANNEXURE II
PROFORMA FOR REGISTRATION OF SUBJECTS FOR
DISSERTATION
1.
Name of the candidate and address
DR. ABHILASHA S. PATIL
(in block letters)
DEPARTMENT OF PERIODONTOLOGY
A.E.C.S. MAARUTI COLLEGE OF DENTAL
SCIENCES AND RESEARCH CENTRE,
BANGALORE-76.
2.
Name of the institution
A.E.C.S. MAARUTI COLLEGE OF DENTAL
SCIENCES AND RESEARCH CENTRE,
BANGALORE-76.
3.
Course of study and subject
MASTER OF DENTAL SURGERY (MDS)
PERIODONTOLOGY.
4.
Date of admission to course
31 st May 2012
5.
Title of the topic
EVALUATION
OF
SALIVARY
BIOMARKERS
OF
PERIODONTITIS
AMONG SMOKERS
AND NONSMOKERS.-A NOVEL STUDY.
6.
BRIEF RESUME OF THE INTENDED WORK:
6.1 Need for the study
Periodontitis is a disease characterized by loss of connective tissue attachment and
bone around the teeth in conjunction with the formation of periodontal pockets due to
the apical migration of the junctional epithelium. Although bacteria are obviously the
initiating agent in periodontitis, the complexity of the associated microflora and the
critical role of the host in determining the outcome of the bacterial challenge cause
difficulties in defining specific disease markers in periodontal diseases. Periodontal
disease progression is episodic in nature on a tooth site level;however, the risk of
developing periodontal disease is principally patient-based rather than site-based.1
Bacterial virulence factors either result directly in degradation of host tissues or cause
the release of biologic mediators from host tissue cells that lead to host tissue
destruction.Important class of molecules in tissue destruction is the variety of enzymes
produced by periodontal microorganisms.1 Various bacteria-derived enzymes, such as
collagen-degrading
enzymes,
elastase-like
enzymes,
trypsin-like
proteases,
aminopeptidases and dipeptidylpeptidases, are recognized as important participants in
tissue destruction. Host and bacteria-derived enzymes, proteins and other inflammatory
mediators appear to hold great promise as salivary biomarkers for the diagnosis of
periodontal disease.2
During the past two decades, oral fluids have been investigated as an alternative
diagnostic approach. Initially, the focus was on oral fluids emanating from individual
teeth. This fluid,known as GCF, is an inflammatory exudate collected by dentists on
filter paper strips. GCF is typically in low abundance during health, but increases in
quantity and complexity of inflammatory molecules at disease sites. Its use has several
diagnostic advantages as contributing inflammatory mediators and tissue-destructive
molecules associated with periodontitis appear, and can be detected, in GCF. However,
GCF analyses are time consuming by requiring multiple sampling of individual tooth
sites onto filter paper strips (i.e., up to 32 teeth, or via clinical decision preselecting the
teeth to sample). The procedure is labour intensive and somewhat technically
demanding, requiring equipment for calibrating and measuring fluid volumes. Finally,
the assessment of analysis is expensive since each sample must be evaluated
individually and the required assays are laboratory based and generally cannot be done
chairside.3
Given some of the problems inherent in sampling GCF, the analysis of salivary
biomarkers offers some advantages. Acquisition of saliva is easy, noninvasive, rapid,
and requires less manpower and materials than GCF. Saliva represents a pooled sample
status at the subject level (as opposed to site or tooth-level). Since levels of salivary
analysis have the potential to reflect current disease activity and severity, this can be
advantageous for providing information used in yes/no decision matrices. Knowledge
of levels of specific salivary biomarkers can, in turn, provide patients and healthcare
practitioners with the ability to determine whether a disease is present, whether
initiation of treatment is needed or if treatment has been successful.3
Alkaline Phosphatase (ALP) is associated with the cell plasma membrane, this
enzyme appears to play a role in the transport of substances from the intracellular
compartment across the cell membrane to the extracellular region.ALP measurements
are good indicators of the metabolic activity of bone, this enzyme could be regarded as
a possible biochemical marker in periodontal disease. It isassociated with alveolar bone
destruction, which is a key feature of periodontal disease.4
Acid Phosphatase (ACP) and Alkaline Phosphatase (ALP) are among the enzymes
most commonly associated with bone metabolism.These enzymes are indicators of
higher levels of cellular damage and their increased activities in GCF are a
consequence of their increased release from the damaged cells of the soft tissues of the
periodontium and are a reflection of metabolic changes in inflamed gingiva.Their
increased activities might be a consequence of destructive processes in alveolar bone in
advanced stages of development of periodontal disease.5
Aspartate Aminotransferase (AST) and Alanine Aminotransferases (ALT)are
enzymes that are normally confined to the cell, and are released into the GCF and
saliva after cell injury and cell death. Their levels in periodontitis are related to the type
of tissue affected by necrosis.Fibroblasts from the periodontal ligament produce
significantly lower levels of aminotransferases than gingival epithelial cells.3
Lactate Dehydrogenase (LDH)is the most useful enzyme for screening of
periodontitis.6LDH are soluble cytoplasmic enzymes used widely in medicine as
diagnostic aids to assess cell death and tissue destruction. The major source of salivary
LDH is probably the oral epithelial shedding cells and so salivary LDH may be
evaluated for identifying possible oral mucosal pathologies.LDH is a marker of cell
death and tissue degradation.7
Creatinine(CRE),Blood Urea Nitrogen(BUN) and Urea(UA)are indicators of a
higher level of cellular damage and their increased activity in GCF and saliva is a
consequence of their increased release from the damaged cell of soft tissues of the
periodontium and a reflection of metabolic changes in the inflamed gingiva.8
Free Hemoglobin(free-Hb) is considered as a potential biomarker.6,9
Neopterin, a metabolite of guanosine, belongs to the class of chemical compounds
known as pteridines and is an early and valuable marker of cellular immunity. It has
been shown to be associated with the initiation and progression of periodontal disease.
Increased neopterin concentrations indicate cell mediated immune activation reflecting
the extent and the activity of the disease.10
Smoking is one of the most significant risk factors with respect to the development and
progression of periodontal disease. In terms of the mechanism through which smoking
influences periodontitis progression, various factors contribute to the deleterious
periodontal effects of smoking, including alteration of both microbial and host response
factors. Smoking affects the human immune system and the cellular and soluble
inflammatory system, smoking exerts effects throughout the cytokine network.11The
increased prevalence and severity of periodontal destruction associated
with smoking suggests that the host bacterial inter action normally seen in chronic
periodontitis are altered result in more aggressive periodontal break down. This
imbalance between bacterial challenge and host response may be due to changes in
composition of the sub gingival plaque with increases in the numbers and/or virulence
of the pathogenic organisms; changesthe host response to the bacterial challenges or
combination of both.12A increase in the levels of biomarkers is seen in smokers with
periodontitis.6,11,13
And hence the need was felt to investigate the levels of salivary biomarkers in smokers
with periodontal disease.
6.2 Review of literature
This study 6 aimed to determine the usefulness of salivary biomarkers for the screening
of periodontal disease and examined the agreement between the results of salivary
enzyme tests and probing depth in smokers and non-smokers. One hundred and eightyseven subjects were recruited, periodontal pocket depth probing was performed and
various enzymes and biochemical parameters in saliva were measured. Salivary LDH
level had the highest sensitivity and specificity. Salivary LDH may be feasible and
useful parameter for the screening of periodontal disease and salivary AST and BUN
appear to be potentially useful for this purpose.
This study 4 described the purpose of the study to illustrate the influence of periodontal
disease on the level of salivary Aspartate aminotransferase, alanine aminotransferase
and alkaline phosphatase. All patients included in the study presented a probing depth
>5 mm, bleeding on probing and alveolar bone loss >40%. Salivary AST, ALT and
ALP activities were measured using DiaSys analysis kits from Diagnostic Systems.
Salivary AST activity in patients with periodontal disease was significantly increased
compared with controls. Salivary ALT activity was not significantly modified in saliva
from patients with periodontal disease compared with the control group. There was a
significant increase in salivary ALP activity in patients with periodontal disease
compared with controls.Periodontal destruction such as periodontal pockets, gingival
bleeding and suppuration are related to higher ALP and AST levels in saliva.
The authors in this study 11 examined the prospective association between smoking and
periodontal disease progression and the effects of smoking on salivary biomarkers
related to periodontitis. 219 of 256 (86%) workers examined at baseline completedPD
measurements and saliva samples were collected. Change in PD was used for
assessment of periodontitis progression when three or more sites displayed an increase
of 2 mm over 4 years.Salivary biomarker levels were determined by real-time
polymerase chain reaction and enzyme assay. Levels of salivary markers including
prostaglandin
E2,
lactoferrin,
albumin,
aspartate
aminotransferase,
lactate
dehydrogenase, and alkaline phosphatase were significantly lower in current smokers
than in non-current smokers. No meaningful differences in the proportions of six
periodontal pathogens were observed between current and non-current smokers.
Smoking may suppress the host-defense system which promotes periodontal disease
progression.
The authors in this study13 determined the association between levels of salivary
thiocynate and enzymes and periodontitis. One hundred thirteen samples of
periodontitis with smokers. Periodontitis in non-smokers healthy smokers and healthy
non-smokers were recruited for the study and salivary thiocynate and asparate
aminotransferase (AST), alanine aminotransferase (ALT) and lactate dehydrogenase
(LDH) levels were analyzed. The results showed that salivary levels of thiocynate and
ALT, AST, LDH were significantly higher in smokers with periodontitis as compared
to others.
The authors in this study14 aimed to determine the salivary enzyme levels of aspartate
aminotransferase (AST), alanine aminotransferase (ALT), and lactate dehydrogenase
(LDH) after scaling and to clarify the influence of interleukin (IL)-1 genotypes on these
enzyme levels. Forty-nine Japanese patients with chronic periodontitis (24 men and 25
women) were enrolled in this study. Measurements of clinical parameters including
probing depth (PD), clinical attachment level (CAL), and bleeding on probing (BOP)
and collections of stimulated whole mixed saliva were performed at baseline and 4
weeks after scaling. After evaluation of salivary AST, ALT, and LDH levels, DNA was
extracted from various cells in whole saliva. IL-1A+4845 G/T genotype was
determined by polymerase chain reaction amplification, followed by enzyme digestion
and electrophoresis. Mean PD, CAL, and BOP values significantly decreased after
scaling. No difference was noted in the decrease in PD, CAL, and BOP after scaling
between the carriers and non-carriers of IL-1A+4845 allele 2.Salivary AST, ALT, and
LDH levels reflect inflammation and destruction of periodontal tissue, suggesting
clinically useful markers following periodontal therapy.IL-1A+4845 alleles did not
influence clinical parameters, they may influence post-scaling values of salivary AST
and ALT.
The authors
10
aimed to assess the relationship between clinical parameters and
concentrations of neopterin within gingival crevicular fluid (GCF) from inflamed
gingiva and periodontitis sites before and after the treatment of periodontitis. Sixty age
(35 to 65 years)and gender-matched (30males and 30 females) subjects were recruited
and divided into the following four groups of 15 subjects each based on gingival index,
Ramfjord periodontal disease index, clinical attachment loss (CAL), and radiographic
parameters (bone loss): healthy (group 1), gingivitis (group 2), mild periodontitis
(group 3), and moderate to severe periodontitis (group 4). A fifth group consisted of the
15 subjects from group 4, 6 to 8 weeks after treatment (scaling and root planing). GCF
was collected from each patient, and the neopterin levels were determined by enzyme
immunoassay. Positive correlations between CAL and GCF neopterin concentrations
were seen. Neopterin levels in GCF may be potentially useful as an indicator of
periodontal inflammation and the host response.
This study12 aimed to determine the activity of these enzymes in saliva among smoker
and non smoker patients with chronic periodontitis, and its relation to the clinical
periodontal parameters (plaque index PI, gingival index GI, pocket depth PD and
clinical attachment level CAL). Salivary samples of chronic periodontitis patients non
smokers and smokers were collected for the study of salivary (AST), (ALT), and
(LDH) levels were analyzed spectrometrically. Statistical analysis revealed highly
significant difference in LDH activity (142±14.63IU/L) between smoker and non
smoker groups with significant positive correlation between the activity of LDH and
GI, PD and CAL among smokers. Also significant positive correlation between AST,
ALT, LDH enzymes and CAL in smokers.The author concluded significant positive
correlation between CAL and all enzymes levels among smokers.
This study
9
determined the usefulness of salivarybiomarkers for predicting the
progression of periodontitis. Eighty-five chronic periodontitis patients were enrolled in
an 18-month longitudinal study. Fifty-seven experienced progression of periodontitis,
indicated at the end of the 18 months by at least one site with >3 mm loss of attachment
compared
with
baseline.The
levels
of
aspartate
aminotransferase,
alanine
aminotransferase (ALT), lactate dehydrogenase, alkaline phosphatase and free
haemoglobin
as
biomarkers
Porphyromonasgingivalis,
were
determined,
and
PrevotellaintermediaandTannerella
the
counts
forsythia,
of
which
represented the periodontal bacteria, in the stimulated saliva. Counts and ratios of P.
gingivalis and P. intermedia were found to be significant predictors of the progression
of periodontitis. To increase prediction accuracy, combination analyses were
performed. The combination of ALT level and the P.gingivalis ratio showed the
highest likelihood. The findings suggested salivary ALT level and the P.gingivalis ratio
may be potential indicators for the progression of periodontitis. Such a salivary test
could be a useful diagnostic tool for predicting periodontal disease progression.
6.3 Objectives of the study
The purpose of the present study is to assess the association between periodontal
disease and smoking using the following salivary biomarkers:
7.

Alkaline Phosphatase (ALP),

Acid Phosphatase (ACP),

Aspartate Aminotransferase (AST),

Alanine Aminotransferase (ALT),

Lactate dehydrogenase (LDH),

Creatinine (CRE),

Blood Urea Nitrogen (BUN),

Urea (UA),

Free-Hemoglobin (f-Hb)

Neopterin
MATERIAL AND METHODS
7.1 Source of data:
Patients will be selected from the Outpatient Department of Periodontology, AECS
Maaruti College of Dental Sciences and Research Centre, Bangalore,Karnataka.
7.2 Method of collection of data:
Subject population:
A total of 64 patients aged between 21-60 years will be included in the study.
Assessment of smoking status will be performed according to the criteria established
by Centre for the Disease Control and Prevention (CDC) through a questionnaire.
They will be distributed into four groups according to periodontal status.
Group I: 16 Patients who are non-smokers with clinically healthy periodontium.
Group II: 16 Patients who are smokers with gingivitis i.e. when bleeding was observed
after periodontal pocket probing.
Group III: 16 Patients who are smokers with moderate periodontitis i.e. when atleast
one probing depth was 4-5 mm, clinical attachment loss:3-4mm
Group IV: 16 Patients who are smokers with severe periodontitis i.e. when atleast one
probing depth was 6 mm or more, clinical attachment loss more than 5mm.
The protocol for all procedures is approved by the Institutional Review Board for
Ethical Clearance of AECS Maaruti College of Dental Sciences and Research Centre
and Sri Jayadeva Institute of Cardiovascular Sciences and Research Centre, Bangalore.
Subjects will be informed prior and a valid informed consent will be taken before
collection of the sample.
Inclusion criteria:

Patients aged between 21-60 years.

Patients who had smoked over 100 cigarettes over a lifetime and were smokers
at the time of examination.

Patients included for non-smokers will be those who had not smoked 100 or
more cigarettes in their lifetime.

Patients included for moderate and severe periodontitis will be according to the
classification of the 1999 American Academy of Periodontology workshop.
Exclusion criteria:

Systemic diseases that could influence periodontal conditions,including
diabetes,immunity disorder and leukemia.

Periodontal treatment or antibiotics taken within the previous 3 months.

Pregnancy and lactating females.
Clinical Examination:Assessment of the periodontal condition of each subject will be made and recordedby a
single examiner using Williams graduated periodontal probe.
Clinical Parameters examined will be:

Gingival Index (GI) ( Loe and Silness,1963)

Bleeding on Probing(BOP) ( Ainamo and Bay, 1975)

Probing Pocket Depth(PPD)-Probing Pocket depth will be measured at six sites
(mesio - facial, mid - facial, disto - facial and mesio -lingual, mid - lingual,
disto - lingual) per tooth for all teeth, excluding the third molars. PPD for each
site will be measured from the crest of the gingival margin to the base of
gingival sulcus.

Clinical Attachment Level(CAL)-CAL will be measured at six sites (mesio facial, mid - facial, disto - facial and mesio - lingual, mid - lingual, disto lingual) per tooth for all teeth, excluding the third molars. CAL for each site
will be measured from the cemento – enamel junction (CEJ) to the base of
gingival sulcus.
Sampling:
A 5-minute stimulated saliva sample will be collected by asking the patient to chew a
piece of paraffin gum to stimulate salivary flow.
A 2.0 ml of stimulated saliva will be collected in a sterile salivary vial between 9.00am
to 12.00 pm.
Saliva samples will be transferred to a refrigerated container at 40C and then will be
transported to the laboratory. Samples will be stored at -80oC until analysis.
The
kits
to
be
used
will
be
ALP
(Roche
Dedicated
System
Packs,
Germany),ACP(Roche Dedicated System Packs, Germany),ALT(Roche Dedicated
System Packs, Germany),AST(Roche Dedicated System Packs, Germany),LDH(Roche
Dedicated
System
Packs,
Germany),CRE(Roche
Dedicated
System
Packs,
Germany),UA(Roche Dedicated System Packs, Germany),BUN(Roche Dedicated
System Packs, Germany),Neopterin(commercially available ELISA kit, GMBH).
Free-Hb will be measured by colorimetric analysis.
Statistical Analysis:
The collected data will be analysed statistically using the following tests:
Chi-square test and ANOVA.
P values <0.05 will be considered significant.
Any other suitable methods will be used at the time of data analysis.
7.3 Does the study require any investigation or intervention to be conducted on
patients or other humans or animals (If so please describe briefly):
Yes

Clinical periodontal examination will be carried out for patients.

Gingival index will be measured in the oral cavity.

Bleeding on probing will be measured in the oral cavity.

Probing Pocket Depth and Clinical Attachment Level will be measured in the
oral cavity.

A 2 ml saliva sample will be collected for assessment of the levels of salivary
biomarkers.
7.4 Has ethical clearance been obtained from your institution in case of 7.3
Yes
7.5 Any trial test has been carried out?
No
8.
LIST OF REFERENCES
1.Ozmeric N "Advances in periodontal disease markers."
ClinChimActa2004; 343 :1-16
2.ZhangL ,Bradley S ,Paulo M. et al"The Clinical value of salivary biomarkers for
periodontal disease. "
Periodontol 2000 2009;51:25-37.
3. Miller C, Foley J, Bailey A et al."Current developments in salivary
diagnostics."Biomark Med 2010;4(1):171-189.
4.Totan A, Greabu T,Totan C and Spinu T. "Salivary aspartate aminotransferase,
alanine aminotransferase and alkaline phosphatase:possible markers in periodontal
diseases? "
ClinChem Lab Med 2006;44(5):612–615.
5.Dabra S, SinghP"Evaluating the levels of salivary alkaline and acid phosphatase
activities as biochemical markers for periodontal disease: A case series. "
Dental Research Journal 2012; 9(1):41-45.
6.Nomura Y, Tamaki Y, Tanaka T et al."Screening of periodontitis with salivary
enzyme tests. "
Jour Oral Sci. 2006;48(4):177-183.
7. Kugahara T,Shosenji Y,Ohashi K. "Screening for periodontitis in pregnant women
with salivary enzymes."
J.Obstet.Gynaceol.Res.2008;34(1):40-46
8.MahmoodM. "Evaluation of the salivary biomarkers creatine kinase (CK) & blood
urea nitrogen (BUN) activities before& after non surgical periodontal treatment in
patients withchronic periodontitis."
J Bagh College Dentistry 2011;48(3):107-111.
9.Nomura Y, Shimada Y,Hanada Net al. "Salivary biomarkers for predicting the
progression of chronic periodontitis."
Arch Oral Biol 2012;57:413-420.
10.Pradeep A.R, Kumar M, Ramachandraprasad M, and ChowdhryS. "Gingival
Crevicular Fluid Levels of Neopterin in Healthy Subjects and in Patients With
Different Periodontal Diseases."
J Periodontol 2007;78:1962-1967.
11.Kibayashi M, Tanaka M, Nishida N et al." Longitudinal Study of the Association
Between Smoking as a Periodontitis Risk and Salivary Biomarkers Related to
Periodontitis."
J Periodontol 2007;78:859-867.
12. Mohammad A. "Salivary enzymes as markers of chronic periodontitis
among smokers and non smokers."
J Bagh College Dentistry 2011;23(3):83-86
13.Rai B, Kharb S, Anand S. "Salivary Enzymes and Thiocynate: Salivary Markers
of Periodontitis among Smokers and Non-smokers; a Pilot Study."
Adv. in Med. Dent. Sci.2007; 1(1): 1-4.
14.Yoshie H, Tai H, Kobayashi Tet al. "Salivary Enzyme Levels After Scaling and
Interleukin-1 Genotypes in Japanese Patients With Chronic Periodontitis. "
J Periodontol 2007;78:498-503.
9.
Signature of candidate
10.
Remarks of guide
11.
Name and
letters)
designation
This is a genuine study which will be
carried out by the post graduate student
under my supervision and guidance.
(in
11.1 Guide
block
DR. V. RANGANATH
PROFESSOR & HEAD
DEPARTMENT OF PERIODONTOLOGY
A.E.C.S. MAARUTI COLLEGE OF
DENTAL SCIENCES AND RESEARCH
CENTRE, BANGALORE-76.
11.2 Signature
11.3 Co – Guide (if any)
DR. RAJESH NAIK
PROFESSOR
DEPARTMENT OF PERIODONTOLOGY
A.E.C.S. MAARUTI COLLEGE OF
DENTAL SCIENCES AND RESEARCH
CENTRE, BANGALORE-76.
11.4 Signature
11.5 Head of department
11.6 Signature
12.
12.1 Remarks of chairman and
principal
12.2 Signature
DR. V. RANGANATH